title: Preparation of reference stocks suitable for evaluation of alternative NAT-based mycoplasma detection methods.
abstract: OBJECTIVE
  The aim of this study was to optimize conditions for preparation and cryopreservation of mycoplasma reference materials suitable to evaluate alternative nucleic acid testing (NAT)-based assays and to compare their limits of detection (LODs) with those of conventional culture-based methods.
  
  
  RESULTS
  Acholeplasma laidlawii, Mycoplasma gallisepticum and Mycoplasma arginini stocks with low ratios of genomic copies to colony forming units (12, 8 and 4, respectively) harvested in early stationary phases of growth were preserved with different cryoprotective agents (CPAs) under slow (1°C min(-1)), moderate (8°C min(-1)), fast (13°C min(-1)) and 'snapshot' (60°C min(-1)) cooling rates. Depending on mycoplasma species, increasing the cooling rate from slow to snapshot enhanced cell survival up to 5-fold. The addition of 10% (v/v) dimethyl sulfoxide (DMSO) and 15% (v/v) glycerol significantly improved cell survival of all tested strains. Cryoprotected stocks maintained high and stable titres for at least 1 year during storage at -80°C. Sonication of cell cultures prior to cryopreservation enhanced cell dispersion and reduced of GC/CFU ratios.
  
  
  CONCLUSIONS
  It is feasible to prepare stable reference stocks of cryopreserved mycoplasma cells suitable to reliably compare NAT- and culture-based mycoplasma testing methods.
  
  
  CONCLUSIONS
  This study describes experimental results demonstrating the preparation and storage of highly viable and dispersed mycoplasma reference stocks suitable for comparing alternative NAT-and conventional culture-based mycoplasma detection methods.
authors.affiliation: Center for Biologics Evaluation and Research, Food and Drug Administration, Rockville, MD, USA.
authors.forename: A
authors.initials: A
authors.lastname: Dabrazhynetskaya
authors.forename: V
authors.initials: V
authors.lastname: Furtak
authors.forename: D
authors.initials: D
authors.lastname: Volokhov
authors.forename: B
authors.initials: B
authors.lastname: Beck
authors.forename: V
authors.initials: V
authors.lastname: Chizhikov
keywords: cell aggregation; cell viability; cooling rate; cryopreservation; detection; mycoplasma contamination; optimization